JP3754958B2 - High magnification microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a high-magnification microscopic observation apparatus, and more specifically, a high-magnification (400 to 20000 times) digital micro microscope, a long-time time-lapse observation microscope, a nanomaterial alignment apparatus, a UV laser micro-cutter (cell trimming chromosome cut), a microscopic object The present invention relates to a high-magnification microscopic observation apparatus that can be suitably used in various fields such as a laser trapping (capturing / moving) apparatus, an egg cell injection apparatus, a DNA chip spotting apparatus, and others (inkjet EL production apparatus, ellipso, near field).
[0002]
[Prior art]
For example, when controlling a stage below a micro, such as a microscope, in order to directly grasp the amount of movement of the stage and the current location, a length measuring device using a magnetic scale, an optical scale, laser interference, etc. is attached to the stage and measured. Position control is almost impossible unless full closed control that feeds back a signal from the long device to the motion control unit is employed.
[0003]
However, even if the stage is precisely controlled, for example, if an error occurs due to a difference from the thermal expansion of the workpiece, it is impossible to say that the fixed position of the workpiece is constantly pressed, even in a fully closed control. Therefore, measures such as using a material having a similar coefficient of thermal expansion or providing a separate environmental sensor for temperature compensation have been taken, but it is complicated as a symptomatic therapy and causes restrictions.
[0004]
In addition, since individual differences of the workpieces naturally occur, it is necessary to set the control position of the stage in accordance with the individual differences of the workpieces, and this is the biggest problem in terms of automation yield. It is not realistic because the work structure itself requires the same accuracy. Furthermore, in a system in which a length measuring device is incorporated on the stage, even if the stage can be precisely positioned, the error cannot be corrected if the observation system moves.
[0005]
If a single-axis drive motor is employed, naturally, an XY biaxial motor is required, and a plurality of (for example, XYΘ) length measuring instruments are inevitably required. For this reason, errors are inevitably accumulated, so that alignment accuracy becomes stricter and cost increases are unavoidable. Therefore, it is ideal to align the desired position of the workpiece while directly observing the workpiece itself. If the position information is given to the control system, it is possible to precisely align the workpiece itself. Is possible. Therefore, an improvement in yield can be expected.
[0006]
Furthermore, when manufacturing highly integrated products such as semiconductors and biochips, the most cost-effective method is to capture the workpiece placed on the stage two-dimensionally. It is going to be adopted actively. Although the method of illuminating the object has been devised to make it easier to confirm the target, there has been no solution with high visibility. In addition, a new database for coding and managing patterns had to be considered.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to use pattern image recognition by attaching a marker or the like having high visibility to a slide glass or a stage of a microscope. That is, an object of the present invention is to realize a highly accurate full-closed control device that does not require a conventional expensive length measurement scale and a workpiece manufactured using the same.
[Means for Solving the Problems]
According to the present invention, in order to solve the above-described problems, a predetermined pattern formed on a sample holder for a microscope or a stage, a recognition unit for recognizing the predetermined pattern, and position information recognized by the recognition unit And a moving means for moving the microscope sample holding material to a predetermined position, a high-magnification microscopic observation apparatus is provided.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the microscope sample holding material is, for example, a microscope slide glass, on which a predetermined pattern such as a code or a marker is formed. This predetermined pattern is, for example, a mark formed by irradiating a slide glass with a laser or the like during observation under a microscope, a chrome deposited film, a screen printed film, or a photosensitive material marked with a UV or YAG laser, or erased. Electronic paper or EL material that has been engraved or laser irradiated so that it can be rewritten, such a light-emitting body that has been coated with a code or shape displayed on it, or a probe or pen that is under pressure like electronic paper Can be mentioned, and the part is marked, but is not limited thereto. The pattern itself can also be formed by a light emitting element.
[0010]
Light-emitting elements are electrically, chemically, optically or magnetically excited to produce a liquid crystal that produces an electro-optic effect, colloidal particles used in electronic paper, fluorescent electroluminescent elements, and phosphorescence. An element that displays a spot of light by an element, a projector element such as a laser spot or an optical shutter array can be exemplified, but the invention is not limited thereto.
[0011]
The shape of the pattern itself is not particularly limited, but for example, a lattice shape or a staggered shape is preferable. Furthermore, if a predetermined pattern is formed on a slide glass or the like and placed on a light emitting element such as a liquid crystal panel, the liquid crystal panel or the like can be used as a light source, and the slide glass or the like can be used as an opening array.
[0012]
When liquid crystal is used as the light-emitting element, a plurality of pixels are arranged, and a liquid crystal display panel having a width of 5 to 500 μm, preferably 10 to 100 μm can be used. If the thickness is less than 5 μm, the influence of the interference of the diffracted light cannot be ignored by the Fraunhofer rule as in the case of the diffraction grating, and this becomes disturbance light and the precise position cannot be specified. This is because the linearity characteristic of the moving position versus the output is distorted. Furthermore, the pitch is 5 to 500 μm, preferably 10 to 100 μm. This is for the same reason as the pixel width described above.
[0013]
In the case of a liquid crystal display panel, a pair of transparent substrates are opposed to each other, and liquid crystal is sealed between them. Examples of the transparent substrate include a glass substrate and a polyacrylic substrate, but are not limited thereto. In addition, for example, an ITO film is preferably used as the transparent electrode film, but is not limited thereto. The transparent electrode film is formed by vapor deposition using a CVD method or the like.
[0014]
Furthermore, the liquid crystal may be, for example, a birefringent liquid crystal element, a transmission / scattering liquid crystal element, a TN (twisted nematic) liquid crystal, an STN (super TN) liquid crystal, a ferroelectric liquid crystal element, an antiferroelectric liquid crystal, or a polymer dispersed liquid crystal. However, the present invention is not limited to these.
[0015]
The distance (cell gap) between the opposing substrates is 1 to 100 μm, preferably 1 to 20 μm.
[0016]
As the recognition means, a known photodetector, for example, a charge coupled device (CCD) camera, a discharge device (CID) camera, a video camera, a photomultiplier tube, a parallel vision chip or the like can be used. It is not limited to these. In addition to the direct reading by the recognizing means, the reading is formed on the optical reading head by applying a material using phase change that can be read and written, for example, like a DVD read head. A method of reading pits may be used. Alternatively, the detection unit may be a magnetic head coated with a magnetic material on the slide glass and the pits may be read by the same method. In addition, an IC card, a flash memory, or the like may be mounted and formed on a glass and read by an electronic reading device.
[0017]
Moreover, as a moving means, the combination with other actuators, such as a linear motor, a stepping motor, a piezo, and an ultrasonic motor, can be used, for example. In addition, the microscope apparatus that is the subject of the present invention includes all microscopes such as an optical microscope, an AFM / STM microscope, and an electron microscope.
[0018]
According to the present invention, a pattern such as a code or a marker is engraved on a slide glass or the like of a microscope, and pattern matching detection is performed by a CCD or the like, and feedback is performed to an actuator to perform positioning. By identifying the points, the workability of sample observation can be improved. Since the code of the slide glass is detected with an optical magnification of 10 to 50 times, the position in the slide glass can be specified with a resolution of 100 nanometers to 1 micron as a resolution.
[0019]
As for this code, a pattern may be created in advance on a slide glass as a ready-made product. When the microscope image is observed, the printed shape and characters may be displayed on the slide glass. Not only positioning patterns but also past observation data can be stored in memory. Moreover, the method of carrying out inkjet printing on a slide glass may be used.
[0020]
Furthermore, according to the present invention, a predetermined pattern formed on the microscope sample holding material, means for recognizing the predetermined pattern, a processing apparatus for processing the sample held on the microscope sample holding material, There is provided a high-magnification microscopic observation apparatus including a moving means for moving the microscope sample holding material or the processing apparatus to a predetermined position based on position information recognized by the recognition means. As a result, positioning between the sample processing apparatus and the sample can be performed at high speed.
[0021]
Here, examples of the processing apparatus for processing the sample include a manipulator, a laser cutter, a laser marker, and the like, but are not limited thereto. Any processing machine represented by a micro factory can also be used. The processing device is preferably installed at the above-described means for recognizing the predetermined pattern.
[0022]
Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a configuration of a high-magnification microscopic observation apparatus according to the present invention. In FIG. 1, reference numeral 1 denotes a positioning XY stage, which can be moved in the XY direction by an actuator such as a piezoelectric motor (not shown). A slide glass 2 is placed on the XY stage 1, and a specimen sample S is placed on the slide glass 2 and covered with a cover glass (not shown). The microscope barrel 3 is disposed at an approximate position of the specimen sample. The microscope barrel 3 accommodates an optical system such as an objective lens and a condenser lens, a photoelectric element for imaging, and the like. The image observed with the microscope is displayed on the monitor 4.
[0023]
Further, the microscope barrel 3 is provided with a detector 5 which is branched and made of a CCD, and this detector 5 is arranged on an approximate position of a positioning marker which will be described later. The image of the detector 5 is displayed on the monitor 4 as a multi-window, and the signal enters the positioning controller (CPU) 6 for position recognition processing. A predetermined movement amount is calculated by the position recognition process, and the positioning XY stage 1 is moved by an actuator (not shown).
[0024]
The state of the slide glass 2 on the positioning XY stage 1 is shown in FIG. 2A is a top view and FIG. 2B is a side view. The same components as those in FIG. The slide glass 2 is disposed in contact with three positioning pins 9 provided on the stage 1, and the specimen sample S is covered with a cover glass (not shown) by a vacuum chuck 10.
[0025]
On the inner surface of the slide glass 2, a positioning marker 7 is formed of a chromium vapor deposition film 12 or the like. The marker 7 forms a pattern as shown in FIG. 3 and has a 15 μm square opening 11. A liquid crystal display module 8 is bonded to the positioning marker 7, and specific lighting light of the liquid crystal display module 8 is transmitted through the opening 11. This makes it possible to light any position of the positioning marker 7.
[0026]
The liquid crystal display module 8 is configured by sandwiching liquid crystal between an upper substrate and a lower substrate. The liquid crystal display module is, for example, a size of 20 mm × 20 mm as a whole, and has 105 dots vertically and 74 pixels horizontally. It is configured. Note that the size of the liquid crystal display dots is one dot assigned to each opening of each positioning marker, and is 200 to 300 micron square. Even if the opening of 15 μ is displaced at the time of attachment, the purpose can be achieved and positioning can be permitted if it is within 200 to 300 μm square. Each pixel can be selectively energized by a lead wire, and the liquid crystal sandwiched between the upper and lower substrates is driven by energization to emit light from a backlight (not shown) installed on the bottom surface of the substrate. By making it transmit and light, the pattern can be lighted.
[0027]
When the position of the sample on the slide glass 2 is specified, marking on the slide glass 2 in this way facilitates the installation of the slide glass 2. Even when high accuracy is required, the ease of the installation is does not change. Arbitrary positions between the openings where the absolute coordinates on the slide glass 2 can be specified can be divided by the number of pixels by the detector 5 which is a CCD.
[0028]
As a positioning method, the shape of the opening 11 can be stored in advance as a pattern by a pattern matching method, and the position can be specified in increments of pixels depending on which coordinates on the image the center of gravity of the shape is located. become. In other words, the pattern of the opening 11 expressed on the image (CCD image) composed of 640 × 480 pixels captured by the detector 5 and the position appearing at the center of gravity are recorded as observation points, and the observation position is specified again. In this case, the actuator is controlled to reproduce the image. The slide glass 2 may be formed directly on the upper surface of the glass panel of the liquid crystal display module 8 below the slide glass 2 by vapor deposition / etching without forming such an opening.
[0029]
The position specifying method is performed based on the flow of FIG. The slide glass 2 is set on the positioning XY stage 1, the sample, that is, the specimen sample S is observed by the microscope barrel 3, and the measurement point is set. In the detector 5 integrated with the lens barrel 3, the specific opening 11 of the positioning marker 7 on the slide glass 2 enters the visual field.
[0030]
At that time, the liquid crystal display module 8 is sequentially turned on as shown in FIGS. That is, first, pixels in one column direction are displayed, and the columns are sequentially scanned (FIGS. 5A to 5C). When entering the field of view of the detector 5, the column is sequentially turned on by one pixel (liquid crystal display unit dot) in units of rows (FIG. 5 (d) to FIG. 6 (f)). As a result, the position of a certain opening in the visual field of the detector 5 is specified from the address of the liquid crystal display. The opening in the visual field of the detector 5 stores a pattern in advance as shown in FIG. 7G, and determines where the center of gravity position is in the visual field as coordinates on the image.
[0031]
Since the pattern storage allows the size and shape of the opening to be recognized as a pattern, as shown in FIG. 7G, by selecting the opening periphery and the opening in advance, the CPU recognizes the figure as a figure. For example, when the position is determined by further dividing one liquid crystal dot by 640 × 480, the area to be selected for registration as pattern information is about 30 × 30 to 300 × 300 pixels in 640 × 480 pixels. Choose to be. The size of the area to be selected is determined according to the size of the opening. When it is 60 × 60 or less, the pattern matching accuracy is lowered and the positioning accuracy is deteriorated. When the pixel size is 300 × 300 pixels or more, the effective area that can be actually detected on the image is reduced, which causes a non-measurable region in the entire liquid crystal panel. The most effective size is about 60 × 60 to 100 × 100 pixels.
[0032]
This makes it possible to narrow down the position on the slide glass over a wide range. Since the position can be specified from the address of the liquid crystal and the coordinates on the image, if the address and the coordinates on the image are stored in memory, the position can be reliably reproduced.
[0033]
【The invention's effect】
According to the present invention, even if the slide glass is once removed, it is possible to easily determine the observation position again at the 100 nano level. Further, even if the stage, the slide glass, or the lens barrel is displaced due to temperature change, vibration, or the like, the position can be quickly identified. Further, if the image is continuously recognized and observed in a state where pattern matching is performed, the correction is performed autonomously in real time, so that a long time-lapse observation can be easily performed. If a high-speed parallel processing image sensor or the like is used, an active vibration isolation function of about 1 to 10 kHz can be realized.
[0034]
Moreover, this invention is applicable not only to a slide glass but to various positioning workpieces. Similar positioning is possible if such a marking is printed in advance when the workpiece is finely positioned and the marking is placed on the liquid crystal display. That is, even when moving from the processing apparatus to the processing apparatus, if the address of the liquid crystal and the coordinate information on the image are transmitted, the positioning can be performed easily and accurately.
[0035]
In addition, in a system in which a length measuring device is incorporated on a conventional stage, even if the stage can be precisely positioned, if the observation system or the like moves, the error cannot be corrected. Since the stage is captured from the observation unit as an image, the entire optical system is very stable. In particular, if a manipulator or the like for processing a sample is installed at the observation unit, positioning between the manipulator and the sample can be performed live.
[0036]
Moreover, since the autonomous correction stage can be performed by feeding back the image information of the aperture array using the light emitting element, it is possible to actively correct the positional deviation when there is a temperature fluctuation or vibration. This eliminates the need to install a microscope on a vibration isolator such as a vibration isolation table or to perform observation or production processing in an environment adjustment room that suppresses changes in wind and temperature. By using this, if a high-magnification ultra-deep microscope, a long-time fluorescence observation lime lapse microscope, or the like is constructed, a clearer image can be obtained. Furthermore, if a high-speed image pickup device such as a parallel vision chip is used, the visual feedback response speed is remarkably increased, so that disturbances such as fluctuations, vibrations and sounds can be completely removed. In the future, by using a high-speed response imaging element such as a parallel vision element, higher frequencies can be removed.
[0037]
In particular, in the assembly process, the load in the work environment such as micro and nano is reduced, so a prototype device for research and development (equipped with a laser cutter, a laser marker, a manipulator, etc.) and an inspection device for production. Also, it can be applied to a processing machine represented by a micro factory. When these features are combined, according to the present invention, a disturbance prevention function, a length measuring microscope function, a high-precision positioning function, and the like are possible. In particular, in the conventional microscope, a function of performing distance measurement in the visual field of the microscope by image processing or the like is added, but distance measurement including outside the visual field of the microscope can also be performed.
[0038]
Further, while observing a moving object with a microscope using image recognition or pattern matching according to the present invention, it is possible to simultaneously measure and analyze the moving distance and locus using a liquid crystal panel. That is, when the measurement object moves during long-time fluorescence observation, etc., the distance of the movement is determined in the process of autonomously moving the stage position so that the object remains in the field of view by pattern recognition. If the absolute coordinates are read in time series in the same manner on the liquid crystal panel, even if the object moves in a wide range, the movement distance can be measured in time series with high resolution.
[0039]
Furthermore, it can be replaced with a slide glass DNA chip or the like. In that case, such a code is embedded next to the DNA array carved on the slide glass. Accordingly, in order to increase the reading accuracy in the case of fluorescence identification or increase the throughput, it can be used as a precision positioning chip when a method alternative to the conventional image processing comes out.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of a high-magnification microscopic observation apparatus according to the present invention.
2A is a top view of a positioning XY stage, and FIG. 2B is a side view of a positioning XY stage.
FIG. 3 is a diagram showing a positioning marker.
FIG. 4 is a diagram illustrating a position detection method.
FIG. 5 is a diagram illustrating (a) to (e) among diagrams (a) to (g) illustrating a method of specifying scanning and position of a liquid crystal display.
FIG. 6 is a diagram showing (f) among diagrams (a) to (g) showing a method for specifying scanning and position of a liquid crystal display.
FIG. 7 is a diagram showing (g) among the diagrams (a) to (g) showing a method for specifying the scanning and position of the liquid crystal display.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Positioning XY stage 2 ... Slide glass 3 ... Microscope barrel 4 ... Monitor 5 ... Detector 6 consisting of CCD ... Positioning controller (CPU)
7 ... positioning marker 8 ... liquid crystal display module 9 ... positioning pin 10 ... vacuum chuck 11 ... opening 12 ... chrome vapor deposition film S ... specimen sample

Claims (8)

Based on a predetermined pattern formed on the microscope sample holding material, a recognition means for recognizing the predetermined pattern, and position information recognized by the recognition means, the microscope sample holding material is moved to a predetermined position. And a high-magnification microscopic observation apparatus. The high-magnification microscopic observation apparatus according to claim 1, wherein the predetermined pattern is formed by a light emitting element. The high-magnification microscopic observation apparatus according to claim 1, wherein the predetermined pattern is formed by a light emitting element and an opening array. The light-emitting element is composed of a liquid crystal that generates an electro-optical effect when excited electrically, chemically, optically or magnetically, and colloidal particles used in electronic paper, an electroluminescent element, an organic electroluminescent element, or a light-emitting diode. An element combined with a light source, or an element that displays a light spot by a colloidal particle used in electronic paper, an electroluminescence element, an organic electroluminescence element or a light source alone comprising a light emitting diode, or a projector element comprising a laser spot or an optical shutter array The high-magnification microscopic observation apparatus according to claim 2 or 3. Recognized by the predetermined pattern formed on the microscope sample holding material, the recognition means for recognizing the predetermined pattern, the processing apparatus for processing the sample held on the microscope sample holding material, and the recognition means A high-magnification microscopic observation apparatus comprising: a moving means for moving the microscope sample holding material or the processing apparatus to a predetermined position based on position information. 6. The high-magnification microscopic observation apparatus according to claim 5, wherein the processing apparatus is a manipulator, a laser cutter, a laser marker, a spotter, or an STN / AFM probe. 7. The high-magnification microscopic observation apparatus according to claim 1, wherein the recognition means is a charge coupled device (CCD) camera, a discharge device (CID) camera, a video camera, a photomultiplier tube, or a fast response imaging device. 8. A high-magnification microscopic observation apparatus according to claim 1, wherein the moving means comprises a linear motor, a stepping motor, or a combination of these with an actuator comprising a piezo or ultrasonic motor.

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